Removal of residual cell culture impurities

ABSTRACT

The present application discloses methods for removing residual impurities from protein preparations. Such methods include addition of an anionic detergent to a solution comprising proteins of interest and cellular contaminants under non-precipitating conditions and passing the solution through an ion exchange column.

RELATED APPLICATIONS

This international patent application claims priority to U.S.Provisional Application 61/904,747 filed Nov. 15, 2013 and EuropeanApplication 13199257.0 filed Dec. 20, 2013, the contents of each ofwhich are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to production of proteins in host cells andimproved purification methods thereof.

BACKGROUND OF THE INVENTION

Various methods for producing vaccines and other biologics in cellcultures have been pre-described. If continuous cell lines are used forthe production, there is the risk that residual DNA of the cell linecould be oncogenic. It is therefore required to destroy and removeresidual DNA from therapeutic proteins of interests. For viral vaccinesthe FDA currently recommends a DNA amount of less than 10 ng/dose and afragment size of less than 200 base pairs (Guidance of Industry.Characterization and Qualification of Cell Substrates and otherBiological Materials Used in the Production of Viral Vaccines forInfections Disease Indications. FDA/CBER February 2010; downloadableunder:http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/vaccines/ucm202439.pdf).

Several methods for removing residual DNA from cell culture derivedvaccines have been described. U.S. Pat. No. 5,948,410 describes a methodfor producing flu vaccines derived from cell culture in which a DNAsetreatment is combined with a splitting step using CTAB. WO 2007/052163describes a method for producing flu vaccines derived from cell culturein which beta propiolactone (BPL) is used to inactive the virus and todegrade the residual DNA. Afterwards, the virus is split, e.g., bytreatment with CTAB. The fragmented DNA is then removed from the viruspreparation. Nevertheless, there is still the need to further improveremoval of residual cellular DNA from influenza virus preparation orfrom other products of interest produced in continuous cell lines.

The use of caprylic acid in combination with ion exchange chromatographyfor the removal of cellular DNA from antibodies produced in cell culturehas been disclosed in US 2012-0101262. However, US 2012-0101262 requiresthe use of caprylic acid under conditions that induce precipitation ofresidual DNA and contaminating proteins (in particular at low pH).Afterwards, the precipitate and the protein of interest can beseparated, and the latter is further purified via ion exchangechromatography.

Various other methods of removing impurities and aggregates derived fromcell cultures using caprylic acid or caprylate salts have been describedin the art. Steinbuch (Steinbuch, M. et al. Arch. Biochem. Biophys.134:279-94 (1969) describes recovering IgG from human plasma bycaprylate precipitation of nonenveloped and enveloped viruses therein.U.S. Pat. No. 7,553,938 describes purification of antibodies from astarting solution by adding caprylate or heptanoate ions at pH 4.6 toabout 4.95 and filtering the solution through at least one anionexchange resin. U.S. Pat. No. 5,886,154 describes a process forpurification of antibodies from human plasma involving suspension ofantibodies at pH 3.8 to 4.5 followed by addition of caprylic acid at pH5.0 to 5.2 to precipitate contaminating proteins and lipids while theantibodies remain in solution. The use of caprylic acid is employed inantibody purification because short fatty acids form insoluble complexeswith alpha and beta globulins and at acidic pH whereas the gammaglobulins are not as readily precipitated (Chanutin et. al., 1960). Thusthe gamma globulin can easily be separated. Yet, none of thesedisclosures teach or suggest using an anionic detergent to removeresidual DNA from viral proteins under conditions which preventprecipitation as taught herein.

SUMMARY OF THE INVENTION

The present invention relates to manufacturing of proteins and improvedpurification methods thereof. In particular, the invention providesmethods for removing cellular contaminants, such as residual nucleicacids, from protein products produced in a suitable host (e.g., hostcells). Accordingly, the invention also encompasses related compositionsprepared by such methods.

The invention thus includes methods, which increase the yield, purityand/or safety of biological products produced from cell culture.Biological products prepared by methods of the invention may include,but are not limited to: biopharmaceuticals, proteins, polysaccharides,viral antigens, and antibodies. In a particular aspect, the methodprovides a biological product substantially free of residual DNA.

The inventors have surprisingly found that purification of samplecomprising protein and DNA derived from cell culture is significantlyimproved by an addition of an anionic detergent to a solution comprisingthe proteins and cellular DNA, followed by a purification stepcomprising an ion exchange matrix. The problem to be solved might relateto the inefficiency of separation of negatively charged DNA impuritiesfrom proteins of interest on a positively charged ion exchange matrix.It can be assumed that secondary interactions were playing a role in thediminished ability of anion exchange chromatography to adsorb thenegatively charged DNA impurity. The present invention now achieves anenriched product and increased yield thereof, by contacting residual DNAwith an anionic detergent solution and processing the residual DNA byadsorption over an anion exchange matrix. The inventors haveunexpectedly found that the present invention also removes influenzanucleoproteins. Efficient removal of contaminants as achieved by thepresent invention allows higher yields of product, because incubationand infection times can be increased so that a higher amount of theprotein of interest can be obtained. The invention also encompasses therecognition that a particularly effective removal of residual DNA frominfluenza viruses produced in cell culture can be achieved, if the viruspreparation is purified via an ion exchange chromatography in thepresence of an anionic detergent, such as fatty acid detergents (e.g.,sodium caprylate).

Surprisingly, the inventors have also found the process described hereinsubstantially removes the influenza Nucleoprotein (NP). Advantageously,the invention is not restricted to influenza virus derived from cellculture, but is also applicable to influenza viruses produced in eggs,if removal of NP is desired.

Unlike methods described in prior art (see the “Background” above), thepresent invention employs an anionic detergent under conditions that donot precipitate the proteins of interest or DNA. According to theinvention, the preparation of the protein of interest is separated fromcontaminating DNA/proteins through ion exchange chromatography. By usingthe process described herein, the amount of impurities (e.g., residualDNA) in the sample can be dramatically reduced. This invention can beused to remove residual cellular DNA from any samples containing one ormore proteins of interest produced in host cells, such as cell culture.

Accordingly, the present invention provides a method for removingresidual cellular DNA from a sample comprising a protein of interestproduced in host cells, such as cell cultures, comprising adding ananionic detergent to a solution comprising the protein of interest undernon-precipitating conditions and passing the solution through an ionexchange matrix to remove residual cellular DNA. The methods of theinvention are not limited by a particular protein of interest.Non-limiting examples of proteins that can be purified in accordancewith the present invention include, but are not limited to: therapeuticproteins, antigens (e.g., immunogenic proteins), antibodies or fragmentsthereof.

According to the invention, a suitable starting material which can besubjected to the methods provided herein may be a solution comprising aprotein of interest. Such solution may be a crude cell or tissuepreparation, a partially purified preparation, culture media in whichcells were grown, or cell culture supernatant, etc., but is likely tocontain residual cellular contaminants desired to be removed.

The protein of interest may be grown in a suitable host cell system andcan be purified or clarified from cell impurities by common separationtechniques known in the art. Optionally, further steps may be takenprior to the passage of protein through the ion exchange matrix,preferably prior to the addition of the anionic detergent. For examplethe protein of interest may first be purified from cell cultureimpurities to produce a solution which has been clarified. The eluate orflow-through obtained from the method of the present invention can besubjected to further processing steps, such as purifying the protein ofinterest and formulating it into a vaccine. In some embodiments of theinvention, the anionic detergent is added to the clarified solution bycontacting the solution comprising the protein of interest and cellculture impurities with an anionic detergent solution undernon-precipitating conditions and passing the solution through an ionexchange matrix. Non-precipitating conditions are conditions under whichno substantial precipitation or proteins or DNA occurs.

Thus, the present invention is suitable for the production of viralproteins. Viral proteins of interest may be produced in a suitable host(such as cultured cells) infected with the virus. In some embodiments,the process of viral protein production may include splitting ofvirions, which typically involves the use of a splitting agent oranother detergent. In some embodiments, the anionic detergent used inthe methods described herein is not the splitting agent or the detergentused in the splitting process.

Alternatively or additionally, the invention provides a method fordecreasing residual cellular DNA by passing a solution comprisingproteins, cellular DNA in the presence of an anionic detergent throughan ion exchange matrix under non-precipitating conditions and adsorbingsubstantially all of the cellular DNA on the ion exchange matrix. In apreferred aspect the anionic detergent is not the splitting agent oranother detergent used in process. Further steps may be taken prior tothe passage of proteins and cellular DNA through the ion exchangematrix, preferably prior to the addition of the anionic detergent. Forexample the virus may be split with a splitting agent and the proteinsmay be separated from cell culture debris comprising the split virus toproduce a solution which has been clarified. The eluate or flow-throughobtained from the ion exchange matrix produced by the present inventionmay be subjected to further processing steps such as further purifyingthe viral protein and formulating it into a vaccine.

The present invention is in particular applicable for the preparation ofviral proteins for vaccine production. In another embodiment the presentinvention provides a method for removing residual cellular DNA from asample comprising viral protein produced in cell culture, comprisingadding an anionic detergent to a solution comprising the protein ofinterest under non-precipitating conditions, passing the solutionthrough an ion exchange matrix, whereby the residual cellular DNA isbound to the ion exchange resin. In a preferred aspect the anionicdetergent is not the splitting agent or another detergent used inprocess. Optionally further steps may be taken prior to passage throughthe ion exchange matrix, preferably prior to the addition of an anionicdetergent. For example the virus may first be split with a splittingagent followed by separation of the split virus from cell culture debristo produce a solution which has been clarified. The eluate orflow-through obtained from the ion exchange matrix produced by thepresent invention may be subjected to further processing steps such asfurther purifying the viral protein and formulating it into a vaccine.

A particularly effective purification method for biological productsderived from cell culture should make it possible to optimally removeimpurities such as host cell DNA, while at the same time achieving amaximum yield of product. To this end, the present invention providesproducts substantially free of impurities and enriched for theimmunogenic protein. According to the invention, residual DNA andimpurities derived from host cells such as cell culture propagation maybe removed from the intended product by passage in a solution comprisingan anionic detergent, which is subsequently processed through an ionexchange matrix.

Accordingly, the present invention provides a method for preparing avaccine composition comprising proteins of interest derived from a cellculture comprising adding a fatty acid detergent (as defined below) to asolution comprising proteins of interest under non-precipitatingconditions and processing the protein of interest on an ion exchangematrix. The present invention may be useful for biopharmaceuticalvaccine products.

In a preferred aspect, the invention provides a method for producing aninfluenza vaccine composition comprising immunogenic proteins derivedfrom a virus derived from cell cultures comprising adding a fatty aciddetergent to a solution comprising immunogenic proteins undernon-precipitating conditions and processing the immunogenic proteins onan ion exchange matrix. The immunogenic proteins include hemagglutinin,neuraminidase, and nucleoproteins obtained from an influenza virus whichhas been subjected to inactivation and splitting agents. Additionalsteps may be taken prior to processing the immunogenic protein on theion exchange matrix, preferably prior to the addition of a fatty aciddetergent. For example the influenza virus may first be split with asplitting agent followed by separation of the split virus from cellculture debris to produce a solution which has been clarified. Theeluate or flow-through obtained from the ion exchange matrix produced bythe present invention may be subjected to further processing steps suchas further purifying the viral protein and formulating it into avaccine. In a preferred aspect the fatty acid detergent is not thesplitting agent or another detergent used in process.

As mentioned above, the present invention also encompasses thesurprising finding that the process described herein substantiallyremoves influenza nucleoprotein (NP). Advantageously, the invention isnot restricted to influenza virus derived from cell culture, but is alsoapplicable to influenza viruses produced in eggs, if removal of NP isdesired.

Thus, the invention provides a method for removing viral nucleoproteinsfrom viral proteins of interest. An anionic detergent is added to asolution comprising viral nucleoproteins under non-precipitatingconditions. In some embodiments, the anionic detergent is not thesplitting agent or another detergent used in process. The nucleoproteinscan then be bound to an ion exchange matrix to produce an eluate (orflow-through) comprising the proteins of interest which aresubstantially free of viral nucleoproteins and cellular DNA. In someembodiments, a suitable anionic detergent solution used for the presentinvention does not include deoxycholate, sodium lauryl sulfate, orcombination thereof.

Accordingly the present invention provides a method for removinginfluenza nucleoproteins from an influenza virus preparation derivedfrom cell culture or embryonated eggs comprising adding a anionicdetergent to the virus preparation under non-precipitating conditions,and processing the virus preparation through an anion exchange matrix,whereby the nucleoprotein is bound to the anion exchange matrix.Additional steps may be taken prior to processing the virus preparationon the ion exchange matrix, preferably prior to the addition of ananionic detergent. For example the influenza virus may first be splitwith a splitting agent followed by separation of the split virus fromcell culture debris to produce a solution which has been clarified. Theeluate or flow-through obtained from the ion exchange matrix produced bythe present invention may be subjected to further processing steps suchas further purifying the viral protein and formulating it into avaccine. In a preferred aspect the anionic detergent is not thesplitting agent or another detergent used in process.

The present invention provides an influenza vaccine produced by themethod of the present invention which is substantially free of residualDNA, and nucleoprotein. The influenza vaccine can be formulated in asubvirion particle form, for example HA and NA proteins may be purifiedsubunit proteins or bound to portions of influenza viral structures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a bar graph comparing percent yield of HA proteinprocessed on TMAE and SARTOBIND Q using different chaotropic agents.

FIG. 2 provides a denaturing gel comparing samples from chromatographyruns with and without capryliate as a detergent.

FIG. 3 provides a bar graph comparing ratio of DNA/protein recoveredfrom ion exchange matrices run with different amounts of caprylate.

FIG. 4 provides percent yield of protein recovered from ion exchangematrices run with different amounts of caprylate.

FIG. 5 provides the downstream process for obtaining a cell culturebased subunit influenza vaccine, as described in Onions et al., 2010.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Protein of Interest

The methods of the invention can be used to purify any protein ofinterest derived from a host cell source (such as cell cultures) fromresidual host cell contaminations, such as cellular DNA. Modern virusproduction methods as described here have much in common withbioprocessing of recombinant protein or monoclonal antibody production.Thus, in a particular aspect, the methods are employed to purifyproteins of interest, e.g., therapeutic proteins, immunogenic proteinsor antigens, antibodies or fragments thereof, generated in host cells,such as eukaryotic (e.g., mammalian, avian, insect, plant, fungal, etc.)cell cultures and prokaryotic (e.g., bacterial) cell cultures, celllysates thereof, clarified bulk (e.g., clarified cell culturesupernatant), or animal derived protein mixtures or extracts.

In certain embodiments, the methods comprise effectively removing hostcell contaminants (e.g., impurities) from a mixture (host cell-derivedpreparation, e.g., a cell culture, cell lysate, clarified bulk, etc.)containing one or more proteins of interest. In some embodiments,suitable starting materials for the methods described herein includehost cell-derived preparations (such as sample solutions and celllysates) comprising one or more proteins of interest and residual hostcell contaminants in an amount that is undesirable for intendedpurposes. In some embodiments, such starting materials are crude celllysates. In some embodiments, such starting materials are cell culturesupernatants comprising secreted proteins (for example, cell culturemedia in which host cells are grown). In some embodiments, such startingmaterials are presented as a partially purified form.

Thus one aspect of the present invention provides a method for removingresidual cellular DNA from a sample comprising a protein of interestproduced in a suitable system, such as cell culture, comprising steps ofadding at least one anionic detergent to a solution comprising theprotein of interest under non-precipitating conditions; passing thesolution through an ion exchange matrix, whereby the residual cellularDNA is bound to the ion exchange resin, so as to separate the protein ofinterest from the residual DNA (e.g., in an eluate or flow-through);and, optionally, further purifying the protein of interest andformulating it into a product. In some embodiments, the resultingpurified protein of interest is suitable for use in the manufacture ofpharmaceutical compositions. Thus, such protein or proteins may beformulated as a pharmaceutical product, such as biologic therapeuticsand vaccines.

Accordingly, the methods described herein are useful for the preparationof viral proteins produced in a suitable host. In some embodiments, suchviral proteins are viral immunogenic proteins (i.e., viral antigens)suitable for vaccine production.

Immunogenic proteins suitable for use in the invention may be derivedfrom any virus which is the target of a vaccine. The immunogenicproteins may be formulated as inactivated (or killed) virus, attenuatedvirus, split virus formulations, purified subunit formulations, viralproteins which are isolated, purified or derived from a virus, and viruslike particles (VLPs).

If during vaccine production, a splitting step is to be used, thesplitting agent may be different from the anionic detergent of themethods described herein. Preferably the splitting step or splittingagent is added prior to the ion exchange chromatography on which theresidual cellular DNA is bound or separated from the protein ofinterest.

The immunogenic proteins of the invention are viral antigens whichpreferably include epitopes which are exposed on the surface of thevirus during at least one stage of its life cycle. Viruses may benon-enveloped or, preferably, enveloped. Viruses are preferably RNAviruses, and more preferably ssRNA viruses. They may have a sense or,preferably, an antisense genome. Their genomes may be non-segmented or,preferably, segmented. Preferred viruses of the invention includeinfluenza virus comprising viral antigens such as neuraminidase (NA) andhemagglutinin (HA) proteins.

Virus Culture

The invention provides a method of preparing an influenza virus, andremoval of residual DNA or impurities generated during the processing ofa viral antigens for vaccine production. Accordingly, the inventionprovides a method for removing nucleoproteins from an influenza viruspreparation. Influenza virus may be cultured in a host and purificationsteps taken to isolate and purify NA and HA proteins. Thus in one aspectof the present invention relates to a method for removing InfluenzaNuclear Protein (NP) from a preparation comprising virus proteins ofinterest, comprising splitting a virus preparation obtained from cellculture or eggs, contacting the virus preparation with a anionicsurfactant under non-precipitating conditions and processing thepreparation through an ion exchange matrix, whereby the nuclear proteinis bound to the anion exchange resin, and optionally further purifyingthe viral protein and formulating it into a vaccine.

The culture host may be cells or embryonated hen eggs, which aresuitable for producing a vaccine that can be used for administration tohumans. Non-limiting examples of suitable cells which have been approvedfor vaccine manufacture include MDCK cells, CHO cells, Vero cells andPER.C6® cells. For the embodiments of the inventions involving the useof eggs, the viruses may also be propagated in eggs. The currentstandard method for influenza virus growth for vaccines uses embyronatedSPF hen eggs, with virus being purified from the egg contents (allantoicfluid). It is also possible to passage a virus through eggs andsubsequently propagate it in cell culture and vice versa. Methods forpurification of vaccine products cultivated in embryonated eggs isdescribed, for example, in GB 1498261.

Preferably, the cells are cultured in the absence of serum, to avoid acommon source of contaminants. Various serum-free media for eukaryoticcell culture are known to the person skilled in the art, e.g., Iscove'smedium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences).Furthermore, protein-free media may be used, e.g., PF-CHO (JRHBiosciences). Otherwise, the cells for replication can also be culturedin the customary serum-containing media (e.g., MEM or DMEM medium with0.5% to 10% of fetal calf serum).

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can be used. In some embodiments, the cells maythus be adapted for growth in suspension. The suspension may first beclarified using any method known in the art. The clarification stepserves to remove cells, cell debris, and host cell impurities from thesample. In some embodiments, clarification may be performed via one ormore centrifugation steps. Centrifugation of the sample may be performedby routine methods known in the art. For example, centrifugation may beperformed using a normalized loading of about 1×10⁻⁸ m/s and agravitational force of about 5,000×g to about 15,000×g.

Purification

In another aspect, the suspension may be clarified via one or more depthfiltration techniques. Depth filtration refers to a method of removingparticles from solution using a series of filters, arranged in sequence,which have decreasing pore size. A depth filter three-dimensional matrixcreates a maze-like path through which the sample passes. The principleretention mechanisms of depth filters rely on random adsorption andmechanical entrapment throughout the depth of the matrix. In variousaspects, the filter membranes or sheets may be wound cotton,polypropylene, rayon cellulose, fiberglass, sintered metal, porcelain,diatomaceous earth, or other known components. In certain aspects,compositions that comprise the depth filter membranes may be chemicallytreated to confer an electropositive charge, i.e., a cationic charge, toenable the filter to capture negatively charged particles, such as DNA,host cell proteins, or aggregates.

The methods according to the invention also include harvesting andisolation of viruses or the proteins generated from cell culture. Duringisolation of viruses or proteins, the cells are separated from theculture medium by standard methods such as separation, filtration orultrafiltration. The viruses or the proteins are then concentratedaccording to methods sufficiently known to those skilled in the art,such as gradient centrifugation, filtration, precipitation,chromatography, etc., and then purified. It is also preferred accordingto the invention that the viruses are inactivated during or afterpurification. Virus inactivation can occur, for example, byβ-propiolactone or formaldehyde at any point within the purificationprocess.

Any depth filtration system available to one of skill in the art may beused throughout the steps of present invention. In a particularembodiment, clarification and purification by depth filtration may beaccomplished with a MILLISTAK+Pod depth filter system, X0HC media,available from Millipore Corporation. In another aspect, the depthfiltration step may be accomplished with a ZETA PLUS Depth Filter,available from 3M Purification Inc.

Vaccine Production

Vaccines are generally based either on live virus or on inactivatedvirus. Inactivated vaccines may be based on whole virions, ‘split’virions, or on purified surface antigens. Antigens can also be presentedin the form of virosomes. The invention can be used for manufacturingany of these types of vaccines. It is particularly suitable formanufacturing influenza vaccines, however, which generally compriseresidual DNA and nucleoprotein in a detectable amount. Such influenzavaccines include live virus, whole virion or split virion influenzavaccines. Where the vaccine is formulated in a subvirion form, the viralantigens can be found in a split virus form, where the viral lipidenvelope has been dissolved or disrupted, or in the form of one or morepurified viral proteins.

As a further alternative, the vaccine may include a whole virus, e.g., alive attenuated whole virus, an inactivated whole virus, etc. Methodsfor inactivating or killing viruses to destroy their ability to infectmammalian cells are known in the art. Such methods include both chemicaland physical means. Chemical means for inactivating a virus includetreatment with an effective amount of one or more of the followingagents: detergents, formaldehyde, formalin, BPL, and UV light.Additional chemical means for inactivation include treatment withmethylene blue, psoralen, carboxyfullerene (C60) or a combination of anythereof. Other methods of viral inactivation are known in the art, suchas for example binary ethylamine, acetyl ethyleneimine, or gammairradiation. Preferably, the virus is inactivated with BPL.

Residual DNA may be inactivated with an alkylating agent that cleavesthe DNA into portions small enough so that it is unable to code for afunctional protein. Preferably, the length of degraded residual cellculture DNA is less than 500 base pairs. More preferably, the length ofdegraded residual cell culture DNA is less than 200 base pairs.Preferably, the use of an alkylating agent such as betapropiolactone(BPL) in the invention provides the additional benefit of reducingaggregation and contaminants. Vaccine formulations with reducedaggregates may also have improved immunogenicity. US 2009-0304729teaches the treatment of functional residual DNA with alkylating agents.Prior to the use of the anionic detergent in combination with ionexchange chromatography, parts of the fragmented residual DNA can beremoved by precipitation with a cationic detergent like CTAB asdescribed in Onions et al. (2010; Biologicals, 38(5): 544-551). Thewhole downstream process of Onions is shown in FIG. 5. In someembodiments, the present invention can be applied as part of the Onionsprocess.

Methods of splitting viruses, such as influenza viruses, are well knownin the art, e.g., see International Patent Publications: WO 02/28422, WO02/067983, WO 02/074336, WO 01/21151, etc. Splitting of the virus iscarried out by disrupting or fragmenting whole virus, whether infectious(wild-type or attenuated) or non-infectious (e.g., inactivated), with adisrupting concentration of a splitting agent. Splitting agentsgenerally include agents capable of breaking up and dissolving lipidmembranes, typically with a hydrophobic tail attached to a hydrophilichead. A preferred splitting agent is cetyltrimethylammoniumbromide(CTAB). The disruption results in a full or partial solubilization ofthe virus proteins, altering the integrity of the virus. Preferredsplitting agents are non-ionic and ionic (e.g., cationic) surfactants,e.g., alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines,betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin,lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols(e.g., the Triton surfactants, such as Triton X-100 or Triton N101),polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethyleneethers, polyoxyethylene esters, etc.

One useful splitting procedure uses the consecutive effects of sodiumdeoxycholate and formaldehyde, and splitting can take place duringinitial virion purification (e.g., in a sucrose density gradientsolution). Thus a splitting process can involve clarification of thevirion-containing material (to remove non-virion material),concentration of the harvested virions (e.g., using an adsorptionmethod, such as CaHPO₄ adsorption), separation of whole virions fromnon-virion material, splitting of virions using a splitting agent in adensity gradient centrifugation step (e.g., using a sucrose gradientthat contains a splitting agent such as sodium deoxycholate), and thenfiltration (e.g., ultrafiltration) to remove undesired materials. Splitvirions can usefully be resuspended in sodium phosphate-bufferedisotonic sodium chloride solution.

A composition (such as a vaccine) that is “substantially free ofresidual DNA” refers to a composition or formulation, wherein residualDNA fragments of less than 200 basepairs are detectable at less than 10ng per 0.5 ml, as determined by capillary electrophoresis (see, e.g., WO2009/118420). The total amount of residual DNA in compositions of theinvention is preferably less than 20 ng/ml, e.g., ≦10 ng/ml, ≦5 ng/ml,≦1 ng/ml, ≦100 pg/ml, ≦10 pg/ml, etc.

Accordingly, an assay used to measure residual DNA will typically be avalidated assay (Guidance for Industry: Bioanalytical Method Validation.U.S. Department of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research (CDER) Center forVeterinary Medicine (CVM). May 2001; Lundblad (2001) Biotechnology andApplied Biochemistry 34:195-197). Three principle techniques for DNAquantification can be used: hybridization methods, such as Southernblots or slot blots (Ji et al. (2002) Biotechniques. 32:1162-7);immunoassay methods, such as the THRESHOLD System (Briggs (1991) JParenter Sci Technol. 45:7-12; and quantitative PCR (Lahijani et al.(1998) Hum Gene Ther. 9:1173-80). These methods are all familiar to theskilled person, although the precise characteristics of each method maydepend on various factors such as choice of probes for hybridization,the choice of primers and/or probes for amplification, etc.

In another aspect, the invention provides methods for preparinginfluenza vaccine compositions which have reduced levels ofnucleoproteins (NP). Preferably, NP makes up less than 15% by mass ofthe total influenza virus protein in the vaccine, e.g., <12%, <10%, <8%,<7%, <6%, <5%, <4%, <3%, <2%, or <1%. The vaccine may comprise less than3 μg NP per 10 μg of HA, less than 2.5 μg NP per 10 μg of HA, less than2 μg NP per 10 μg of HA, less than 1.5 μg NP per 10 μg of HA, less than1 μg NP per 10 μg of HA, less than 0.5 μg NP per 10 μg of HA or lessthan 0.1 μg NP per 10 μg of HA. Most preferably, the vaccine issubstantially free of NP. This is understood as having less than 0.1 μgNP per 10 μg of HA. In some embodiments, the methods provided herein mayachieve at least 10-fold reduction in the amount of NP in a preparation,e.g., at least 10-fold, at least 12-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 75-fold, or at least 100-fold reduction in the amountof NP in a flow-through (or eluate) as compared to the starting materialsubjected to the purification methods of the invention.

Methods to determine the amount of protein in a composition are known tothe skilled person in the art. However, since NP and NA have virtuallythe same molecular weight (around 60 kD), they usually co-migrate innon-reducing gels. Classic SDS gel-electrophoresis might therefore notbe an appropriate way to determine the amount of NP (see Chaloupka etal., 1996, Eur J Clin Microbiol Infect Dis. 1996 February;15(2):121-7.). One way to determine the amount of NP in a vaccine bulkmight be a 2 dimensional electrophoresis with a subsequent densitometry.Preferred, however is isotope dilution mass spectrometry using anisotopically labeled synthetic peptide as described, for example, in:Williams et al., Vaccine 30 (2012) 2475-2482. Such method uses liquidchromatography-tandem mass spectrometry (LC-MS/MS) using isotopedilution in conjunction with multiple reaction monitoring (MRM). Thismethod quantifies targeted peptides released by proteolytic digestion ofthe sample as a stoichiometric representative of the analyte protein. Astable isotope-labeled reference peptide is spiked into the sample as aninternal standard (IS). Quantification of NP is achieved by comparingthe peak area of the isotopically labeled reference peptide with that ofthe endogenous target peptide. This method allows simultaneousquantification of multiple proteins, provided labeled peptides areincluded for each specific target.

Alternatively, label free mass spectrometry (LC/MSE) is used for thequantification, preferably in quadrupole time-of-flight (Q-Tof) massspectrometers (Getie-Kebtie et al., (2013): Influenza and OtherRespiratory Viruses 7(4), 521-530). For this method, alternating scansof low collision energy and elevated collision energy during LC/MSanalysis are used to obtain both protein identity and quantity in asingle experiment. Quantification is based on the experimental datashowing that the average signal intensity measured by LC/MSE of thethree most intense tryptic peptides for any given protein is constant ata given concentration, regardless of protein type and size. As thesignal intensity is proportional to concentration, the amount of anyprotein in the mixture can be estimated.

The present invention also includes influenza vaccines based on virusesgrown in cell culture (preferably mammalian or avian cells), whereby thevaccines have an amount of residual cellular DNA of less than 5 ng/dose(e.g., less than 4 ng, less than 3 ng, less than 2 ng or less than 1 ngper dose) at a fragment size of less than 200 base pairs, and wherebythe vaccine contains less than 1 μg NP per 10 μg of HA, less than 0.5 μgNP per 10 μg of HA, or less than 0.1 μg NP per 10 μg of HA. Mostpreferably, the vaccine is substantially free of NP. This is understoodas having less than 0.1 μg NP per 10 μg of HA. In particular theinfluenza vaccine is contains less than 1 ng residual DNA per dose at afragment size of less than 200 base and less than 0.5 μg NP per 10 μg ofHA. This vaccine is most preferably free from mercury-containingpreservatives and antibiotics. The vaccine is most preferably atetravalent seasonal or monovalent pandemic influenza vaccine with anamount of residual cellular DNA of less than 1 ng per dose at a fragmentsize of less than 200 base pairs and less than 0.5 μg NP per 10 μg ofHA.

Such vaccine preparations can be obtained, for example, by the followingprocess, which is a particularly preferred embodiment: A method forproducing an influenza virus vaccine in which the following steps areconducted: Influenza viruses are grown in cell culture, e.g., in MDCKsuspension cells (WO 1997/037000). The viruses are harvested, purifiedand concentrated by 0.45 micrometer filtration and CS chromatography.After addition of detergent (such as polysorbates, e.g., Tween® 80), thevirus preparation is treated with BPL. Afterwards the viruses are splitwith CTAB. After an ultracentrifugation and adsorption step the viralprotein preparation is subject to ion exchange chromatography, usingTMAE or Sartobind Q as a resin. The chromatography is done in thepresence of sodium caprylate (about 50 mM for Sartobind; 100 mM forTMAE) and sodium chloride (400 mM for Sartobind), and 200 mM for TMAE).Afterwards the protein preparation is concentrated by a suitable means,such as ultrafiltration. The proteins might be optionally blended withother virus preparation (in the case of tri- or tetravalent seasonalvaccines), and optionally sterile filtrated, filled and packaged. Theinvention thus includes influenza vaccine obtainable by this process.

It will be evident to the artisan that the measure of the residual hostcell DNA content is not meant as a limitation or defining feature ofthis methodology. Instead, these data in the examples support theessence of the present invention: a large-scale methodology for thegeneration of virus particles that results in a highly purified productthat may be utilized in clinical and commercial settings. It can benoted that the importance of achieving particular DNA levels in thefinal product is product-specific. Viral products produced usingcontinuous cell lines for parenteral use in humans will require the moststringent purity standards but, even in that case, the goals may varyfrom 100 pg per dose up to 10 ng per dose (WHO Requirements for the Useof Animal Cells as in vitro Substrates for the Production of BiologicalsRequirements for Biological Substances No. 50), WHO Technical ReportSeries, No. 878, 1998) or higher, and are likely to be adjusteddepending on the product's indication.

Detergents

The anionic detergents used in the present invention are detergentswhich are added as an extra substance for carrying out ion exchange.Accordingly, the detergent itself is not removed by the ion exchangeprocess nor precipitates the substances processed through the matrix butserves to interact with the hydrophobic regions of the residual DNAand/or the virus or viral proteins, particularly the HA subunit. In someembodiments, the anionic detergent used for the present inventionexcludes deoxycholate and/or sodium lauryl sulfate.

In preferred embodiments, one or more anionic detergents are used. Inpreferred embodiments, fatty acid detergents are used (as definedbelow). In particularly preferred embodiments, eight-carbon fatty acidsare used. For example, in some embodiments, caprylic acids (e.g., sodiumcaprylate) are used.

In one aspect, an anionic detergent solution is added to a solutionhaving the proteins of interest. If used for viral preparations, theanionic detergent is preferably added following inactivation orsplitting of viruses, whereby inactivation may be performed before orafter splitting steps. In one aspect, the anionic detergent, is addedduring or prior to an ion exchange step. The addition of a anionicdetergent significantly improves clearance of residual DNA by at least10%, 20%, 30%, 40% or 50%, as compared to clearance of residual DNAwithout treatment. List of commercially available anionic detergents canbe found, e.g., under:http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14572921.

Preferred anionic detergents are cholates, deoxycholates,1-decanesulfonates, and lauryl sulfate. Other suitable detergentsinclude cetylpyridinium bromide, alkyl benzyldimethylammonium chloride,tetradecyltrimethylammonium chloride, hexadecylammonium chloride, andorinthinyl-cysteinyl-tetradecylamide.

In some embodiments, suitable anionic detergents are fatty aciddetergents. In the context of the present disclosure, fatty aciddetergents are understood to be salts of fatty acid, particularlycarboxylic fatty acids selected from C4-C18 carbon chains, preferablyC6-C10 carbon chains, e.g., C6, C8 and C10. Preferably, the fatty acidsare linear and saturated. In some embodiments, suitable fatty aciddetergent is sodium caprylate or a similar salt of caprylic acid. Asdescribed herein, the addition of caprylate (sodium caprylate) (C8) atneutral pH has been shown to improve protein recovery and preventsprotein aggregation or nonspecific binding. In certain embodiments, thefinal concentration of caprylate acid solution comprising a protein orproteins of interest (such as antibodies or fragments thereof, antigens,therapeutic proteins, toxins, peptides, etc.) has a suitableconcentration of detergent is between 25 mM and 300 mM, preferredbetween 50 mM and 250 mM, particularly preferred between 75 mM and 200mM. The concentration might be about 25 mM, about 50 mM, about 75 mM,about 100 mM, about 125 mM, about 150 mM, about 175 mM, 200 mM about 250mM or about 300 mM, depending on the ion exchange resin orchromatography conditions. A skilled person in the art will be abledetermine the most suitable fatty acid detergent and empiricallyelucidate the concentration of fatty acid detergents to make a solution.For example, it is known that carboxylic acid detergents having lowercarbon chains will have less detergent characteristics, while highercarbon chains will have reduced solubility. Typically, higher detergentconcentration is seen as providing a more robust process acrossdifferent strains of influenza, due to disruption of any hydrophobicinteractions and higher reduction of impurities as illustrated in FIG.2. However, it is important that the amount of fatty acid detergents bepresent in an amount and at a pH to prevent precipitation of theproteins and residual DNA in solution.

In another aspect, the pH of the solution comprising proteins ismaintained at a pH at which no (or an insignificant amount of)precipitation occurs of the proteins, nucleoproteins and residual DNA.For example, for caprylic acid, this is neutral pH. The optimum pHrequired to prevent protein precipitation can readily be determinedempirically by the skilled person in the art. Preferably, the final pHof the mixture should be maintained to be between about 7.0 and 9.0. Insome embodiments, the final pH of the mixture is maintained betweenabout 7.2 and 7.5, e.g., between about 7.2-7.4, between about 7.2-7.3,between about 7.3-7.5, between about 7.4-7.5. In some embodiments, thefinal pH of the mixture is maintained at greater than or equal to about7 (such as between about 7-9, e.g., about 7.0, about 7.5, about 8.0,about 8.5, about 9.0, etc.). In some embodiments, the pH of the solutioncomprising the proteins, residual DNA and caprylate should not bereduced to about 6.0 or less (e.g., about 5, 4, and 3). The pH can beadjusted before and/or after the addition of an anionic detergent (e.g.,caprylate) to the sample. In some embodiments, the pH of the mixturecould be adjusted before the addition of an anionic detergent (e.g.,caprylate). In general, any art-recognized acids or buffers can be usedto alter or adjust the pH of a mixture, including, for example,phosphate- and tris-containing buffers.

The method of the present invention may also be applied to partiallypurified protein samples to further remove DNA or undesired impuritiesby contacting the mixture with an anionic detergent solution underconditions which prevent precipitation of the proteins in the mixtureand passing the mixture through an ion exchange matrix. The methods ofthe invention effectively remove host cell DNA contaminants to aconcentration of <10 ng DNA per dose as recommend by WHO for continuouscell lines and nucleoprotein to a concentration of less than 0.5 μg NPper 10 μg HA. In a particular aspect, the amount of nucleoproteinremoved by the present invention is at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 50%, 60%, 70%, 80% and 90% as determined by SDS-PAGE.

The composition comprising proteins and residual DNA in a solution of ananionic detergent is further processed to recover the desired product.Residual DNA is better adsorbed on an anion exchange membrane in thepresence of the anionic detergent. Surprisingly, influenzanucleoproteins are also captured on the anion exchange membrane asidentified by the inventors by electrophoretic analysis of adsorptionpools. The inventors identified that nucleoproteins run with theresidual DNA when contacted with a solution of anionic detergent. Thisfinding has not been shown before and results in an enriched influenzaproduct which is substantially free of residual DNA.

Accordingly, after residual host cell contaminants are removed bytreatment of the contaminant-containing sample (e.g., cell culture andclarified bulk mixtures) with an anionic detergent and subsequentpurification step in accordance with the methods described herein, suchsample can contain no more than about 10000 ng/mg (e.g., no more thanabout 10000, 5000, 1000, 500, 200, 100, 50, 25, or 10 ng/mg) of proteincontaminants. In some embodiments, such protein contaminants include nomore than about 10000 ng/mg nucleoproteins, e.g., no more than about10000, 5000, 1000, 500, 200, 100, 50, 25, or 10 ng/mg nucleoproteins.

Thus, any influenza product which comprises residual DNA andnucleoprotein can be enriched for HA and NA proteins by contact with ananionic detergent solution and processed through an ion exchange matrix.The person skilled in the art will be able to apply the methods of thepresent invention to influenza products generated from cell culture oregg culture.

Chromatography

The present invention may be used in commercial scale processingtechniques that utilize ion exchange chromatography to produce bulkquantities of the finished product. It is known that during large scalemanufacturing, the effect of the binding affinity between the residualDNA and virus particles or viral proteins may be further compoundedduring the concentration of the virus particles because the DNA maybecome physically trapped during the aggregation of the virus particles.Once the DNA is bound specifically or nonspecifically to the virus, orotherwise entrapped by aggregates of the virus or proteins, the use ofion exchange matrices as described in the art becomes relativelyineffective as a means for efficiently removing the DNA. Accordingly,the present invention relates to a purification process to removeresidual DNA by purification with an anionic detergent solution and/or asuitable concentration or ionic strength provided by a salt buffer overa chromatography matrix.

An anionic Q membrane chromatography capsule may comprise a Mustang Qmembrane a chromatography capsule (available from Pall Corporation) orSartobind Q (a strongly basic anion exchanger membrane, available fromSartorius Stedim Biotech GmbH). Any positively charged ligand attachedto the solid phase suitable to form the anionic exchange resin can beused, such as quaternary amino groups. Commercially available anionexchange resins include DEAE cellulose, POROS. PI 20, PI 50, HQ 10, HQ20, HQ 50, D 50 from Applied Biosystems, SARTOBIND. Q from Sartorius,MONO Q, MINI Q, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE. FASTFLOW, Q SEPHAROSE high Performance, QAE SEPHADEX. and FAST Q SEPHAROSE(GE Healthcare), WP PEI, WP DEAM, WP QUAT from J. T. Baker, HYDROCELLDEAE and HYDROCELL QA from BioChrom Labs Inc., UNOSPHERE Q, MACRO-PREPDEAE and MACRO-PREP High Q from Bio-Rad, Ceramic HyperD Q, ceramicHyperD DEAE, TRISACRYL M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSILLS, QMA SPHEROSIL M and MUSTANG Q from Pall Technologies, DOWEX FineMesh Strong Base Type I and Type II Anion Resins and DOWEX MONOSPHERE77, weak base anion from Dow Liquid Separations, INTERCEPT Q membrane,MATREX CELLUFINE A200, A500, Q500, and Q800, from Millipore, FRACTOGELEMD TMAE, FRACTOGEL. EMD DEAE and FRACTOGEL EMD DMAE from EMD, AMBERLITEweak strong anion exchangers type I and II, DOWEX weak and strong anionexchangers type I and II, DIAION weak and strong anion exchangers type Iand II, DUOLITE from Sigma-Aldrich, TSKgel Q and DEAE 5PW and 5PW-HR,TOYOPEARL SUPERQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and650C from Tosoh, QA52, DE23, DE32, DE51, DE52, DE53, EXPRESS-Ion D andEXPRESS-Ion Q from Whatman.

Chromatographic separation over the ion exchange matrix is operated inflow-through mode. The specific methods used for the chromatographycapture step, including flow of the sample through the column, wash, andelution, depend on the specific column and resin used and are typicallyprovided by the manufacturers or are known in the art.

In an alternative aspect, modulation of ionic strength may also beemployed during the chromatography step. The ionic strength of buffersolution may be determined from both molar concentration and chargenumbers of all the ions present in the solution. The ionic strength, I,may be calculated using following formula:

$I = {\frac{1}{2}{\sum\limits_{i = 1}^{n}\; {c_{i}z_{i}^{2}}}}$

where c_(i) is the molar concentration of ion i (mol·dm⁻³), z^(i) is thecharge number of that ion, and the sum is taken over all ions in thesolution. Generally a 1:1 electrolyte such as NaCl, the ionic strengthis equal to its molar concentration, while multivalent ions contributemore to the ionic strength in the solution, for example, the ionicstrength of the 2:2 electrolyte MgSO₄ is four times that of NaCl.

The preferred ionic strength will optimize the balance between removingthe unwanted residual DNA while maintaining a high viral or proteinyield that retains the antigenicity of the virus in a cost effectivemanner.

The person skilled in art will be able to design a chromatographicseparation program depending on, for example, sample characteristics,chromatograph matrix properties and efficiency of fractionation. Asaline buffer is preferably provided at or near a neutral pH such asabout 7.0. 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 and 7.8. The pH should notbe reduced below about 6 as the proteins of interest may lose theiractivity, aggregate or precipitate in the presence of an anionicdetergent (e.g., fatty acid detergents). Suitable concentrations ofbuffer (e.g., sodium chloride buffer) may be between about 100 mM and1M, such as 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700mM, 800 mM, 900 mM, and 1M. The optimum salt concentration depends onthe ion exchange chromatography resin that is used. The person skilledin the art can easily determine the optimum salt concentration byroutine test. For a TMAE resin the best sodium chloride concentration isabout 300 mM, for SARTOBIND Q it is greater than 400 mM (for the use ofcaprylate as detergent; see Examples below).

As used herein the term “chromatography” refers to the process by whicha solute of interest, e.g., a protein of interest, in a mixture isseparated from other solutes in the mixture by percolation of themixture through an adsorbent, which adsorbs or retains a solute more orless strongly due to properties of the solute, such as pI,hydrophobicity, size and structure, under particular bufferingconditions of the process. In a method of the present invention,chromatography can be used to remove contaminants after the precipitateis removed from a mixture, including without limitation, a cell cultureor clarified cell culture supernatant.

The term “impurities” as used herein generally refers to residual hostcell DNA, empty viral particles, aggregated proteins or matter otherthan the intended component(s) of a product.

“Processing” or “processed” used in the context of the invention refersto a downstream step or steps performed after clarification or theinitial starting materials comprising cellular byproduct and debris,colloidal particulates, large biomass and high cell densities.Techniques used in processing steps include isolation, purification,concentration, centrifugation, filtration, formulation, inactivation,splitting and various analytical operations performed for sterilebiological products. “Processed” also may describe the steps of flowingor passing a sample through a chromatography column, resin, membrane,filter, or other mechanism, and can include a continuous flow througheach mechanism as well as a flow that is paused or stopped between eachmechanism.

Absent explicit teaching, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thus,components can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (e.g., a cation exchange resin) or positively charged(e.g., an anion exchange resin). In one embodiment, the charge can beprovided by attaching one or more charged ligands (or adsorbents) to thesolid phase, e.g., by covalent linking. Alternatively, or in addition,the charge can be an inherent property of the solid phase (e.g., as isthe case for silica, which has an overall negative charge).

Accordingly, the present invention encompasses, but is not limited to,the following embodiments:

1. A method comprising a step of subjecting a first solution containinga protein of interest and an anionic detergent to an ion exchange matrixunder a non-precipitating condition, so as to obtain a second solutioncontaining the protein of interest, wherein the second solution containsless residual cellular contaminants than the first solution.2. The method of embodiment 1, wherein the first solution is selectedfrom the group consisting of: cell or tissue lysates, culture media,cell culture supernatants, plasma, and partially purified proteinsolutions.3. The method of any one of the preceding embodiments, wherein theprotein of interest is selected from the group consisting of:therapeutic proteins, immunogenic proteins (e.g., viral antigens), andantibodies or antigen-binding fragments thereof.4. The method of any one of the preceding embodiments, wherein theanionic detergent is selected from the group consisting of: fatty aciddetergents.5. The method of any one of the preceding embodiments, wherein theanionic detergent is different from any other detergent used in aprocess of protein purification.6. The method of any one of the preceding embodiments, wherein theanionic detergent does not include deoxycholate and/or sodium laurylsulfate.7. The method of any one of the preceding embodiments, wherein the ionexchange matrix comprises a basic anion exchanger membrane.8. The method of any one of the preceding embodiments, wherein thenon-precipitating condition comprises at or near neutral pH.9. The method of any one of the preceding embodiments, wherein thesecond solution is an eluate.10. The method of any one of the preceding embodiments, furthercomprising a step of further purification.11. The method of any one of the preceding embodiments, furthercomprising a step of carrying out sterile filtration.12. The method of any one of the preceding embodiments, furthercomprising a step of formulating the protein of interest into apharmaceutical composition.13. The method of any one of the preceding embodiments, furthercomprising a step of carrying out sterile filtration.14. The method of embodiment 12 or 13, wherein the pharmaceuticalcomposition is a prophylactic composition, therapeutic composition, orcombination thereof.15. The method of any one of embodiments 12-14, wherein thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient.16. The method of any one of embodiments 12-15, wherein thepharmaceutical composition further comprises and adjuvant.17. The method of any one of embodiments 12-16, further comprising astep of packaging the pharmaceutical composition into a sterile closedsystem.18. The method of embodiment 17, wherein the sterile closed system isselected from the group consisting of: vials, syringes, and containers.19. The method of embodiment 17 or 18, wherein the sterile closed systemis plastic or glass.20. The method of any one of embodiments 17-19, wherein the sterileclosed system comprises a siliconized surface.21. A use of the pharmaceutical composition of any one of embodiments12-20, for the manufacture of a medicament for administering a subjectin need thereof.22. The pharmaceutical composition of any one of embodiments 12-20 foruse as a medicament for administering to a subject.23. A method comprising administering to a subject the pharmaceuticalcomposition of any one of embodiments 12-20.24. A viral vaccine comprising no more than 5 ng of residual DNA and nomore than 1.0 μg nucleoprotein per dose.25. The viral vaccine of embodiment 24, comprising no more than 1 ng ofresidual DNA and no more than 0.5 μg nucleoprotein per dose.26. The viral vaccine of embodiment 24, comprising no more than 1 ng ofresidual DNA and no more than 0.1 μg nucleoprotein per dose.27. The viral vaccine of any one of embodiments 24-26, wherein the viralvaccine is an influenza vaccine.28. The viral vaccine of any one of embodiments 24-27, furthercomprising an adjuvant.29. The viral vaccine of embodiment 28, wherein the adjuvant is selectedfrom the group consisting of: alum adjuvants, oil-in-water adjuvants,virosomes and Toll-like receptor (TLR) agonists.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXAMPLES

An H5N1 virus was propagated in MDCK suspension cells, harvested andprocessed as described in Onions et al., 2010. The split viruspreparation was subjected to ion exchange chromatography using aSARTOBIND Q (Sartorius) or a FRACTOGEL TMAE (EMD Millipore) membrane.The optimal salt concentration found for TMAE was determined to beapproximately 300 mM, while the optimal concentration found forSARTOBIND Q was greater than 400 mM. Preparations with differentdetergents and chatoropric reagents were conducted. The pH of the finalcompositions was 7.5, except for the arginine compositions, which had apH of 7.2. DNA reduction was assessed by Picogreen and protein yield wasassessed by the BCA assay. Overall, SARTOBINDQ performed better thanTMAE in DNA reduction; however, all runs utilizing sodium caprylateshowed significant increase in DNA reduction compared to just NaCl.Robust results could be obtained with 50 mM caprylate and 400 mM NaCl ona SARTOBINDQ membrane. BCA values for the arginine might not be exactdue to interference of arginine with the BCA assay. Arginine was notfurther investigated for yield due to insufficiently removing DNA. BCAand DNA data for these conditions are shown in FIGS. 3 and 4.

Samples from the following three runs were further examined by RP-HPLCfor HA content: (i) Control—50 mM phosphate, 300 mM NaCl, pH 7.5; (ii)50 mM phosphate, 100 mM sodium caprylate, 200 mM NaCl, pH 7.5; (iii) 50mM phosphate, 100 mM sodium caprylate, 500 mM NaCl, pH 7.5. These runswere considered to be the best case for the conditions examined. Highercaprylate concentration is seen as providing a more robust processacross strains based on the idea that a higher concentration ofcaprylate would more effectively disrupt any hydrophobic interactionsand overall lead to a higher reduction of impurities. The yields byRP-HPLC were calculated and plotted in FIG. 1.

Material from these three runs was also analyzed by SDS-PAGE and can beseen in FIG. 2. The samples required sample prep prior to running ongels due to low protein concentration. The samples were concentrated 2.5fold to ensure protein concentrations high enough to be visualized bySDS-PAGE. The samples were concentrated using a 15 mL Amicon ULTRA SPINTub with 10,000 MWCO membrane. The adsorption pool was also diluted inbuffer and concentrated 2.5 fold to ensure that low molecular weightcontaminants were not lost during the concentration process. This isdemonstrated by comparing lanes labeled Adsorption and Adsorption,Concentrated. A dramatic difference in purity can be seen by comparingthe control runs and the runs containing caprylate. The nucleoprotein inthe caprylate runs is significantly diminished compared to runs justutilizing NaCl to optimize yield performance.

These experiments have successfully shown that secondary interactions,likely hydrophobic in nature, are playing a role in the diminishedability of AEX chromatography to adsorb the negatively charged impurityDNA. Without wishing to be bound by theory, the addition of caprylate tothe adsorption pool likely disrupts this hydrophobic interaction andallows the binding of DNA and Nucleoprotein to the membrane or resin.

It should be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently features specified in onesection may be combined with features specified in other sections, asappropriate.

Throughout the specification, including the claims, where the contextpermits, the term “comprising” and variants thereof such as “comprises”or “comprising” are to be interpreted as including the stated element(e.g., integer) or elements (e.g., integers) without necessarilyexcluding any other elements (e.g., integers).

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for removing residual cellular DNA from a sample comprisinga protein of interest, the method comprising the steps of: a. adding ananionic detergent to a solution comprising a protein of interest undernon-precipitating conditions; and, b. passing the solution through anion exchange matrix, whereby the residual cellular DNA is bound to theion exchange resin, so as to obtain an eluate comprising the protein ofinterest, wherein the eluate is substantially free of cellular DNA.
 2. Amethod for removing residual cellular DNA from a sample comprising viralprotein, the method comprising the steps of: a. providing a samplecomprising virus grown in a host cell system, wherein the virusexpresses viral protein of interest; b. splitting the virus; c. addingan anionic detergent to a solution comprising the viral proteins undernon-precipitating conditions; and, d. passing the solution through anion exchange matrix, whereby residual cellular DNA is bound to the ionexchange resin, so as to obtain an eluate comprising the viral proteinof interest.
 3. A method for preparing an influenza vaccine compositioncomprising immunogenic proteins derived from a virus propagated on acell culture, the method comprising the steps of: a. growing a virus incell culture, b. adding a fatty acid detergent to a solution comprisingthe immunogenic protein under non-precipitating conditions, and c.processing the immunogenic protein on an ion exchange matrix.
 4. Amethod for removing Influenza Nuclear Protein (NP) from a preparationcomprising virus proteins of interest, the method comprising the stepsof: a. splitting a virus preparation derived from cell culture or eggs,b. adding an anionic detergent to the virus preparation undernon-precipitating conditions, and c. processing the virus preparationthrough an ion exchange matrix, whereby the nuclear protein is bound tothe ion exchange matrix.
 5. The method of claim 1 further comprising aclarification step.
 6. The method of claim 1 further comprising aconcentration step.
 7. The method of claim 1 further comprising a depthfiltration step.
 8. The method of claim 3 further comprising a splittingstep prior to the detergent step (b).
 9. The method of claim 1 furthercomprising an inactivation step prior to the ion exchange step.
 10. Themethod of claim 8 wherein the splitting step comprises treatment withcetyltrimethylammonium bromide.
 11. The method of claim 1, wherein theanionic detergent is not the same as other detergents used in theprocess.
 12. The method of claim 1 wherein the anionic detergent is afatty acid detergent.
 13. The method of claim 1 wherein the anionicdetergent is carboxylic detergent which comprises 4-10 carbons inlength.
 14. The method of claim 13 wherein the carboxylic acid detergentis selected from the group consisting of caprylic(8), valeric(5),caproic(6), enanthic(7), pelargonic(9), capric(10) acid under conditionsin which no precipitation of the viral proteins occurs.
 15. The methodof claim 1 wherein the anionic detergent is present at a concentrationof 25 mM-500 mM.
 16. The method of claim 9, wherein the inactivatingstep comprises inactivation with betapropiolactone.
 17. The method ofclaim 1, wherein the ion exchange resin is Sartobind Q.
 18. The methodof claim 1, wherein the ion exchange resin is Fractogel TMAE. 19.(canceled)
 20. The method of claim 1 wherein the amount of the proteinof interest recovered is at least 90%.
 21. An influenza vaccinecomprising less than 5 ng residual DNA per dose at a fragment size ofless than 200 bases, and less than 0.5 μg NP per 10 μg of HA.
 22. Theinfluenza vaccine of claim 21, further comprising an adjuvant.
 23. Theinfluenza vaccine of claim 22, wherein the adjuvant is selected from thegroup consisting of: alum adjuvants, oil-in-water adjuvants, andToll-like receptor (TLR) agonists.
 24. A method for producing aninfluenza vaccine, the method comprising the steps of: a. splitting avirus grown in host cells, b. adding an anionic detergent to a solutioncomprising viral protein of interest from the virus undernon-precipitating conditions, c. passing the solution through an ionexchange matrix, whereby the residual cellular DNA is bound to the ionexchange resin, so as to obtain an eluate comprising the viral proteinof interest; d. optionally further processing the eluate to provide apreparation comprising the viral protein of interest; and, e. carryingout sterile filtration of the preparation, and optionally filling andpackaging.